Determination of the concentration of an anesthetic in a patient has been of great interest in research and in clinical practice. At present, there are no methods in use that measure the levels of anesthetics in target tissues in real time. As the target tissues take up anesthetics from blood at widely differing rates that depend on the precise condition of the patient, methods that determine the concentration of anesthetics in any one body compartment, such as the lungs or blood, do not reliably reflect the depth of anesthesia of the patient as a whole.
The most reliable method of measuring anesthetics currently in use in surgical procedures is to extract large volumes of blood from the patient for gas chromatography. In the case of infants, so much blood must be removed that the determinations are dangerous to the health of the patient. Moreover, this type of measurement involves significant time delays since the blood sample must be sent to a laboratory for measurement. Obtaining results can take hours or days. Such time delays are not compatible with a surgical procedure on a patient.
The levels of inhalation anesthetics can be monitored with substantially less trauma by measuring the content of such volatiles in the exhaled air by a variety of methods. One device monitors exhaled and inhaled anesthetic levels by the increase in mass of an oil droplet on a piezoelectric crystal that occurs as the anesthetic partitions into the droplet.
The following article is of relevance to the detection of anesthetics in patients: Wolfbeis, O.S., et al. "Fiber Optical Fluorosensor for Determination of Halothane and/or Oxygen," Anal. Chem., 57:2556-2561 (1985). Wolfbeis et al. report the development of a method of monitoring the concentration of halothane in air using a silicone rubber membrane impregnated with a fluorophore that is quenched by this particular type of anesthetic.
Commercially available devices (such as the Datex monitor) monitor certain inhalation anesthetics in air by the intensity of their characteristic infrared absorption spectra.
Other research groups have developed fiber-optic biosensors for analytes not necessarily limited to anesthetics. Reports of this nature, which appear to be relevant to the present invention, are the following:
(1) Krull, U. J., et al. (Krull I), "Towards a Fluorescent Chemoreception Lipid Membrane-Based Optrode," Talanta 35:129-137 (1988).
(2) Krull, U. J., et al. (Krull II), "Supported Chemoreceptive Lipid Membrane Transduction by Fluorescence Modulation: the Basis of an Intrinsic Fibre Optic Biosensor," Analyst, 111:259-261 (1986).
(3) Lakowicz, J. R. et al., "Synthesis and Characterization of a Fluorescence Probe of the Phase Transition and Dynamic Properties of Membranes," Biochemistry, 22:5714-5722 (1983).
Krull I and II report progress in development of fiber-optic based sensors employing fluorescent detection of molecules that partition into lipid layers on the fibers. In these reports, the responses of fluorophores such as 1-anilinonaphthalene-8-sulphonate in "dry" lipid monolayers on optical waveguides to the concentration of gaseous chloroform, hexane, and N, N-dimethyl-aniline are measured. No response to changes in concentration of chloroform and hexane were seen when the fluorophore was embedded in fatty acid monolayers, and only transient changes were observed when the fluorophore was incorporated in phosphatidylcholine/cholesterol monolayers.
The method of determining the concentration of an analyte of the present invention is based on a change in a fluorescence characteristic of a fluorophore embedded in a lipid layer caused by a phase change of the lipid layer. The phase change of the lipid layer is a result of, and proportional to, partitioning of an analyte (such as an anesthetic) into the lipid layer. As opposed to the present invention, Krull I and II do not involve a phase change of the lipid layer as a result of partitioning of the analyte into the lipid layer. Krull I and II involve detection of analytes based on changes in fluorescence intensity of a fluorophore due to changes in fluidity and packing of the lipid layer. Thus, while the method of Krull I and II involves a change within a single phase of a lipid layer induced by an analyte, the present method involves a change between phases induced by the analyte.
Lakowicz et al. describes the synthesis and characterization of a new fluorescence probe for biological membranes. The novel fluorescent probe, 6-palmitoyl-2-[[2-(trimethyl-ammonio)ethyl] methylamino] naphthalene chloride (also known as Patman), consists of a trimethyl ammonium head, a fluorescent naphthalene body, and a hydrophobic palmitoyl tail. Because of its structure, Patman orients itself in a phospholipid membrane with its tail embedded in the core of the lipid bilayer and its head associated with the hydrophilic surface. Patman is described as being a useful probe for studying dynamic properties (fluidity) of biological membranes. Both fluorescence lifetime and emission frequency of the excited state of Patman reflect fluidity changes in the lipid bilayer. Lakowicz et al. does not suggest the use of Patman in a lipid layer which is subject to phase changes due to the presence of an analyte.
In summary, while the above-described references are directed to detection of analytes based on fluorescence measurements and changes, in some cases involving a lipid layer, none of them involve analyte detection or quantitation based on a change in a fluorescence characteristic of a fluorophore based on a phase change in a lipid layer. None of them discuss immobilization of the lipid layer using a hydrogel, another (optional) feature of the present invention which is described hereinbelow. Moreover, none of these references describe or suggest the use of impermeable or semipermeable membranes as barrier layers between the lipid layers and the medium containing the analyte, additional possible features of the present invention which will be summarized and described in detail hereinbelow.
The patent literature related to the present invention is represented by the following patents:
1. Wolfbeis et al., U.S. Pat. No. 4,568,518 PA1 2. Marsoner et al., U.S. Pat. No. 4,657,736 PA1 3. Krull et al., U.S. Pat. No. 4,637,861 PA1 4. Krull et al., U.S. Pat. No. 4,661,235 PA1 5. Miller et al., U.S. Pat. No. 4,666,672
Wolfbeis et al., in U.S. Pat. No. 4,568,518, disclose a sensor element for fluorescence optical measurements, comprising a carrier membrane with fluorescent indicator material immobilized thereon. A network structure containing the indicator material is integrated into a carrier membrane. The carrier membrane is made of cellulose while the network structure permeating the carrier is composed of material containing amino groups, such as hexamethylene diamine or polyethyleneimine and the indicator 1-acetoxypyrene-3,6,8-trisulphochloride.
Marsoner et al., in U.S. Pat. No. 4,657,736, disclose an oxygen sensor element that, when placed in contact with a sample containing oxygen, is capable of indicating the concentration of oxygen in the sample. The sensor element is composed of cured silicone polymer that is permeable to oxygen and a chemically modified oxygen-sensitive fluorescent indicator that is homogeneously embedded in the cured silicone polymer. The fluorescent indicator includes compounds such as polycyclic aromatic hydrocarbons, homocyclic aromatic hydrocarbons, and heterocyclic aromatic hydrocarbons. Usually these fluorescent indicators display fluorescent decay times greater than five nanoseconds. Contact of oxygen with the fluorescent material embedded in the silicone matrix causes quenching of the fluorophore, thereby indicating the oxygen concentration.
Krull et al., in U.S. Pat. No. 4,637,861, disclose an ion permeable lipid membrane-based device capable of detecting a particular chemical species in an aqueous electrolytic solution. The device produces a signal based on increased ion permeability of the membrane when it is exposed to the chemical species to be detected and when an electrical potential is applied across it. Phospholipid molecules are covalently linked to a substrate to produce the lipid membrane of this device.
Krull et al., in U.S. Pat. No. 4,661,235, disclose a lipid based membrane transducer. According to this invention, a lipid bilayer composed of phospholipids and cholesterol is used as a barrier or modulator of ion flow through the membrane. The particular membrane chosen is one that is electrically neutral toward the test solution containing the ion to be measured. A potential is applied across the membrane and conductivity of the ion through the membrane is measured. In this way, the target ionic species can be quantitatively determined.
Miller et al., in U.S. Pat. No. 4,666,672, disclose an "optrode" for sensing hydrocarbons. According to these inventors, a two-component system is employed in the fluorometric detection of halogenated hydrocarbons. A fiber-optic element is used to illuminate a column of pyridine trapped in a capillary tube coaxially attached to one end of the fiber optic. The other component consists of a strongly alkaline solution in contact with the column of pyridine and capped at the other end with a semipermeable membrane. The semipermeable membrane is preferentially permeable to halogenated hydrocarbons and impermeable to water and pyridine. As halogenated hydrocarbon diffuses through the membrane and into the column of pyridine, fluorescent reaction products are formed. Light propagated by the fiber optic from a light source then excites the fluorescent species and fluorescent emission is conducted back through the fiber-optic cable to a detector. The intensity of the fluorescence gives a measurement of the concentration of the halogenated hydrocarbons.
In summary, none of the above-described patents disclose a sensor based on a lipid layer with a fluorophore embedded therein in which a fluorescence characteristic of the fluorophore varies in response to a phase change in the lipid layer caused by an analyte dissolving therein.
More generally, in spite of the above-described background art, a need has continued to exist for new and improved ways of measuring the concentrations of lipid-soluble analytes, such as anesthetics in tissues of patients and animals, in real time.